DESCRIPTION: In smooth muscle, the importance of myosin light chain phosphorylation for force regulation is well established. However, contractions can occur in the absence of a change in the level of myosin light chain phosphorylation and, after reaching a peak during force activation, the level of myosin light chain phosphorylation falls during force maintenance. Thus, a large body of evidence suggests that a regulatory system for force, independent of myosin light chain phosphorylation, may exist. The goal of this grant is to determine the molecular mechanism of force regulation in smooth muscle. This application will test two independent hypotheses. The first hypothesis is that a population of attached crossbridges are present in relaxed smooth muscle, and these attached crossbridges participate in contractions which occur in the absence of a change in the level of myosin light chain phosphorylation. Specific aims for this hypothesis are: 1. to determine the relative population of actomyosinADP cross-bridges present in both rigor and relaxed cells; 2. to determine the mechanical characteristics of actomyosinADP crossbridges; and 3. to determine if actomyosinADP crossbridges participate in contractions which occur without a change in the level of myosin light chain phosphorylation. The second hypothesis is that force maintenance is due to the development of non-cross-bridge force bearing structures. Specific aims for this hypothesis are: 1. to determine the cross-bridge states present during force activation; and 2. force maintenance. To test these specific aims, force and stiffness will be determined in single intact and permeabilized smooth muscle cells activated with either calcium or an agonist. In permeabilized cells, rigor solution, rigor solution containing apyrase, and solutions with low ATP concentration, high ADP concentration, and elevated phosphate concentration will be used to increase the relative population of the actomyosin, actomyosinADP, and actomyosinADPPi cross-bridge states. The relationship between stiffness and frequency of oscillation of each cross-bridge state will be determined using pseudorandom white noise and/or sinusoidal oscillation of muscle length. It will then be possible to determine the cross-bridge states which are: 1. present in relaxed smooth muscle; 2. responsible for contractions which occur without a change in the level of myosin light chain phosphorylation; and 3. present during both force activation and maintenance. During force maintenance, the data will differentiate whether a population of attached dephosphorylated (latch) crossbridges develop, the entire cross-bridge cycling rate slows, a cooperative mechanism leads to the development of a population of slowly cycling dephosphorylated crossbridges, or non-cross-bridge force bearing structures are formed. The studies proposed in this grant application will provide direct evidence to test the roles of both actomyosinADP crossbridges in force regulation, and non-cross-bridge structures in force maintenance. The data will aid in elucidating the mechanism of force activation and force maintenance.